All current retroviral vectors used in human gene therapy protocols have been derived from murine leukemia virus (MLV), an amphotropic C-type retrovirus. However, like all other C-type retroviruses, MLV can only establish an infection in the target cell after one cell division. To bypass this problem, efforts are underway in several laboratories to develop retroviral vectors from lentiviruses, such as the human immunodeficiency virus (HIV-1) the major causative agent of AIDS, which are capable of infecting non-dividing cells. However, serious safety issues make it highly unlikely that such vectors could be used in the clinic.
The present invention describes procedures for the generation and production of safe retroviral vectors derived from C-type retroviruses, which are non-pathogenic for humans and which are capable of transducing genes into specific non-dividing cells. Transduction of genes into non-dividing cells has been achieved in the present invention by using the approaches described herein. Using site-directed mutagenesis, a specific signal (nuclear transportation signal sequence) has been introduced into the matrix protein of the retroviral vector particle. This signal is sufficient to enable the penetration of the nucleus of the non-dividing cell.
The introduction of a nuclear localization sequence into the matrix protein adds a new feature to the C-type retroviral vector particle: the capability to actively penetrate the nucleus of a quiescent cell. This system has two major advantages over current vector systems. First, efficient gene transfer into non-dividing cells is achieved. Moreover, this system is, but does not have to be, combined with an existing cell-type-specific gene delivery system. Second, this system is safe, since the retroviral particles used have been derived from a retrovirus which is non-pathogenic in humans.